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UBC Theses and Dissertations

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UBC Theses and Dissertations

Quantitative measurements of changes in DNA tertiary structures induced by physical and chemical agents Hung, Yip-Chan Jacyln

Abstract

When Chinese hamster V79 lung fibroblast cells are lysed in solution containing sodium chloride, a non-ionic detergent Triton-X-100, and the DNA intercalating dye propidium iodide, intact loops of DNA can be visualized under the fluorescence microscope as a 'halo' region surrounding the periphery of the nuclear matrix. The size of these halos has a biphasic dependence on the concentration of propidium iodide, suggesting that the DNA is organized into supercoiled loops constrained by attachments to the nuclear matrix. However, the induction of strand breaks in the loops of DNA by ionizing radiation results in loss of supercoiling and a uniform halo size regardless of the concentration of propidium iodide. The goal of this project was to use mathematical procedures and image processing techniques to measure the change in the size of the DNA halo. We suggest that such a quantitative assay will provide a fast and objective means to directly measure the effect of physical and chemical agents on the DNA loops. The image acquisition, processing, and segmentation procedures that are necessary for the development of this quantitative assay are performed with the Fluorescence Image Processing System (FIPS). A gradient-threshold algorithm is implemented to segment the image, which in this case is to separate the DNA halo from the background and nuclear matrix. Damage inflicted by two of the better characterized agents, x-radiation and platinum complexes, are used as a test system for this assay. The results reported here suggest that this assay can detect damage to the DNA loops at a radiation dose of as low as one Gray. The size of the DNA loop is calculated to be about 102µm. The biphasic dependence of the halo size on the concentration of propidium iodide is consistent with published data. Studies on the effect on platinum drugs on DNA loops show a difference in the kinetics of the unwinding and rewinding of the supercoils and the size of the halos at these phases also differ for both cis- and trans-diamminedichloro-platinum(II) treated cells. With our increased knowledge of the genome organization, an objective quantitative assay that can provide some insight to the damage and repair of DNA at the tertiary level of DNA organization should prove to be useful.

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